Parasiticide for animals

ABSTRACT

PARASITICIDE FOR ANIMALS WHICH IS MIXED IN A FODDER AND CONTAINS AS AN ACTIVE INGREDIENT A NEW ANTIBIOTIC DESTOMYCIN A WHICH IS PRODUCED BY CULTIVATION OF STREPTOMYCES RIMOFACIENS DEPOSITED UNDER ATCC NO. 21066.

nited States Patent "w 3,715,431

Patented Feb. 6, 1973 The titration equivalent measured in an aqueoussolution is 182. The optical rotation is [a] ='-|7 (C2, in water). Theultraviolet absorption spectrum (in an aqueous Yasushi Yusa Yokohama-shiNaotoshi Akai Kokubunjishi, Shinichi Kondo, Yokohamwshi Kimio Satoh,solution) does not show characteristic absorption at 210 Kawasakbshi,and Yasuharu Sekizawa Tokyo, Japan, 5 400 111,12. but shows only endabsorption. Destomycin A 3,715,431 PARASITICIDE FOR ANIMALS assignors toMeiji Seika Kaisha, Ltd. Tokyo Japan is soluble in water and loweralcohols and hardly soluble N0 Drawing Filed July 7, 1967, 651,713 orinsoluble in common organic solvents. The R, value Claims priority,application Ja a J l 19 of the destomycin A when chromatographed on afilter ll/44,178; Mar. 10, 1967, 42/14,717 paper is 0 by usingwater-saturated n-butanol, 0.96 by Int. Cl. A61k 21/00 10 1.5% aqueousammonium chloride solution, 0.07 by US. Cl. 424-181 5 (31311115phenol-water (3:1), 0.07 by acetone-water (1:1), 0.04

by n-butanol-methanol-water (4:1:2), 0 by benzenemethanol (4:1), 0.20 byt-butanol-acetic acid-water ABSTRACT OF THE DISCLOSURE (2:1:1),0.55-0.62 by 80% aqueous methanol containing Parasiticide for animalswhich is mixed in a fodder and 15% Sodium Chloride d gives only one spotrespectivecontains as an active ingredient a new antibiotic destoyfvalue y thin layer chromatography on active mycin A hi h i d d b l i i fStrgptomyces bon using methanol:% N sulfuric acid (1:4) as a solventrimofaciens deposited under ATCC No. 21066. gives single spots at 0 whenbioautographedt Destomycin A gives positive reaction to ninhydrin,anthrone, Ehrlich reaction and after hydrolysis, red tetra- The presentinvention relates to parasiticides for ani- Z01i11m tion. mals, inparticular to the parasiticide for animals cony -flp sulfonate ofdestomycin A is taining a new antibiotic destomycin A as an active in-White needle crystals and decomposes at r di t, The elemental analysisis as follows: C: 51.67%, H:

It was commonly well known that in the body of do- 621%, N! 352%, S1749%, 01 calcdmestic animals there exist several parasites which exertfor 2o 37 3 13 11 1o 3) 2 C H a harmful influence upon the growth of thedomestic 574%, N! 3. St O: 30.36%.) animals and cause bleeding of thedomestic animals. Destomycin A reacts in methanol with acetic anhydrideAs the result of our numerous searches for antibiotics to give whitecrystals of N-acetyl destomycin A which which exhibit anthelminticactivity we have found that 30 decomposes gradually at 240-260 C. Theelemental the antibiotic destomycin A which is produced by cultureanalysis as follow: C: 46.36%, H: 7.11%, N: 6.14%,

of Streptomyces rimofaciens or its variant exhibits excel- O: 39.39%.(Anal. calcd. for lent effectiveness for stamping out parasites in thebody of the domestic animals. The present invention is based C H N O-3(CH CO)-H O upon the above discovery. 35

Streptomyces rimofaciens is a new strain which was C: 46.49%, H: 6.75%,N: 6.2%, O: 40.50%.) isolated by the inventors from a soil samplecollected When destomycin A is hydrolysed with hydrochloric at Mitsuikepark, Tsurumi-ku, Yokohama city and has acid or sulfuric acid, threemain decomposition products been deposited in American Type CultureCollection unmay be obtained. The first-product is C H N O which der theATCC Number 21066. is a derivative of deoxystreptamine and which has oneDestomycin A is accumulated mainly in the cultured amino and oneN-methyl group. The optical rotation is broth by culture of abovementioned strain in a liquid [a] =-18 (C2, H O). It is dilferentiatedfrom a deculture medium of pHS 69 containing nutrients such ascomposition product of hygromycin B hyosamine carbon sources, organicand inorganic nitrogen sources and the like which are usually utilizedfor producing the C H N 0 known antibiotics, at a temperature of 27-28C. for

2-4 days under submerged aeration condition. (hydmchlondei D Journal oforganlc To recover destomycin A from the cultured broth many Chemlstrlf!27, 2793: The sefiond p f 18 means which are generally applied on theknown anti taloseas 1n hygromycin B and the thlrd product is a kind i imay be 1 The destomycin A may be of amino acid, molecular formula ofwhich is C H NO tracted and recovered in the highest yield from the cul-AS the result of our Structural y. We have detertured broth byadsorption and elution with ion exchange mined the chemical structure ofdestomycin A a resin, particularly cation exchange resin of carboxylic,5"- r hy roXy- "-(1"+amino-2"-hvdroxy acid type.methyl)-tetrahydropyran 2" ylidene}-fl-'D-talopyrano- The new antibioticdestomycin A is white crystals and syl] 1methylamino-3-amino-l,2,3-trideoxymyo-inositoldecomposes gradually at180190 C. The elemental (CZUH37N3OI3).

2" $11 01 NHCH analysis of destomycin A is as follows: C, 44.70%, H:Destomycin A which is an active ingredient of the 7.42%, N: 7.73%, 'O:39.41%, N-CH 3.06%, amino parasiticide for animals of this invention maybe peroral- N; 4,67%, (Anal. c lcd. for C H N O -H O; C; lyadministered. In this case, destomycin A may be ad- 44.03%, H: 7.21%, N:7.70%, O: 41.06%, N-CH (1): ministered in its original powder form as itis or destomy- 2.76%, amino N(2); 5.14%, molecular weight; 545,536). einA is mixed in advance with suitable fodder material such as soybeanmeal, bran and just on administration the mixture will be compounded inthe fodder.

The amount of destomycin A to be mixed with the fodder is preferably1-50 g. per one ton of the fodder. If the fodder containing destomycin Ain above mentioned proportion will be daily administered to the domesticanimals it is observed that the numbers of the parasite eggs in theexcrement gradually decrease and :finally become nil or almost nil.However, no change is noticed on gain of the weight of the body, intakeamount of the fodder, breeding rate and the like and no other secondaryactions are observed.

The anthelmintic activity of destomyin A will be shown by way of thefollowing experimental examples.

EXPERIMENTAL EXAMPLE 1 The fodder mixed uniformly with destomycin A inthe rate of 10 g. per one ton of the former was daily administered toseven head of about three-month-old pigs on which Ascaris suilla andTrichuris suis being parasitic. The excrements from each pig werecollected just before administration and on each 10th day, 20th day,30th day, 40th day and 60th day after administration and the numbers ofparasite eggs in 1 g. of the excrement were measured by Stolls method.The measured results were shown in the following table from which theanthelmintic activity could be confirmed. In addition, the increase ofthe weight of the body and intake amount of the fodder were normal andno other secondary actions were observed.

EXAMPLE 3 Recovery of destomycin A from Streptomyces rimofaciensStreptomyces rimofaciens cultured on a glucoseasparagine agar slantmedium was inoculated to a liquid medium (pH 7.0) containing 2% glucose,1% peptone, 0.3% meat extract, 0.05% K HPO and cultured at 28 C. for 48hours under submerged aeration condition to give a seed culture. 5% ofsaid seed culture was inoculated to 12 l. of a liquid medium (pH 7.2),containing 2.5% glucose, 3.5% soybean meal, 0.5% soluble vegetativeprotein, 0.25% table salt and 0.4% soybean oil, in a 20 1. volume glassfermentor and cultured at 28 C. for 65 hours under submerged aerationcondition. The cultured medium was filtered using Hyflo supercel (Johns-Manville Corp.) as a filter-aid to give 9.5 l. of filtrate (pH 6.4, 450,u/ml.) The filtrate was applied to a column filled with 300 ml. ofAmberlite IRC 50 (NH type) (Rohm & Haas Co. cation exchange resin ofcarboxylic acid type). The column was washed with water and eluted with2% aqueous ammonia. The 300 ml. of the active elute was concentratedunder reduced pressure to dryness to give a crude brown powder (400/mg.). 2.2 g. of the powder was dissolved in 10 ml. of water and appliedto a column :filled with 80 ml. of Dowex 1X2 (OH- type) (50-100 mesh,anion exchange resin, Dow Chemicals Incorp.) and developed with water.The eluate was fractionated to each 10 m1. Destomycin A was eluted in15-24th tubes.

Numbers of parasite eggs discharged Before administration 10th day 20thday 30th day th day 60th day No. of pigs ASE TSE ASE TSE ASE TSE ASE TSEASE TSE ASE TSE No'rE.ASE: Ascuris suilla egg; TSE: Trichuris suie.

EXPERIMENTAL EXAMPLE 2 The fodder mixed uniformly with destomycin A inthe rate of 12 g. per one ton of the former was daily administered tofive breeding hens on which Ascaris galli being parasitic. The crementsfrom each hens were collected just before administration and on each 2ndweek, 4th week, 6th week and 10th week after administration and thenumbers of parasite eggs in 1 g. of the crement were measured by Stollsmethod. The measured results were shown in the following table. Furtherno residual Ascaris galli in the small intestine of the five hensslaughtered after 10th week experiment was observed. From thisexperiment the anthelmintic activity of destomycin A could be confirmed.In addition, the breeding rate and intake amount of the fodder werenormal and no other secondary actions were observed.

The 16-20th fractions of destomycin A were combined and lyophilized togive white powder of destomycin A. The fractions of destomycin A werecombined and applied to a column filled with 5 g. of active carbon,washed with 200 ml. of water and eluted with /s N sulfuric acid. Theactive eluate was adjusted to pH 4.0 with Amberlite IR 45 (OH- type) andlyophilized to give 330 mg. of white powder (890 ,u/mg.) of destomycin Asulfate. Yield was 61 mg. per 1 l. of the cultured filtrate.

We claim:

1. An anthelmintic process for decreasing parasites in domestic animalscomprising feeding a mixture of destomycin A in an amount between 1-50gm. per ton of fodder to said animals.

2. The process of claim 1 wherein the fodder is fed to the animals up to60 days.

3. The process of claim 1 wherein the amount of destomycin A mixed withthe fodder is 10 g. per ton of fodder.

4. The process of claim 1 wherein said parasites are Ascaris suilla,Trichuris suis or Ascaridia galli.

5. The process of claim 4 wherein said animals are hens or pigs.

References Cited Kondo et al., J. of Antibiotics, Ser. A, XVIII-I,January, 1965, pp. 38-42.

Goldberg: Antibiotics, D. Van Nostrand Co., Inc., Princeton, NJ., 1960,pp. 178-9.

JEROME D. GOLDBERG, Primary Examiner

